HILIC Chromatography: When and How?

Importance of the Topic

Hydrophilic interaction liquid chromatography (HILIC) provides enhanced retention and resolution for polar and ionized analytes that may be poorly separated by reversed-phase LC. It offers compatibility with mass spectrometry and expands analytical capabilities in fields such as biopharma, environmental testing, and food safety, where detection of highly polar compounds is critical to quality control and advanced research.

Objectives and Overview

This document presents an overview of HILIC principles, criteria for choosing HILIC versus reversed-phase methods, and best practices for developing robust HILIC assays using Agilent InfinityLab Poroshell 120 HILIC columns. Key topics include method scouting, mobile phase design, column equilibration, sample solvent management, and strategies to protect metal-sensitive analytes.

Methodology and Instrumentation

HILIC retention relies on a water-enriched layer on polar stationary phases that enables liquid–liquid partitioning and ion exchange. Typical workflows use high organic mobile phases (>50% acetonitrile) with volatile buffers (formate, acetate) at pH adjusted for analyte ionization. Major instrumentation components include:

• Agilent 1260 Infinity Binary LC, 1260 Infinity II Binary HPLC, 1290 Infinity II systems
• Triple quadrupole and Q-TOF mass spectrometers (e.g., 6470, 6490, Ultivo TQ/MS)
• Diode array, evaporative light scattering, and fluorescence detectors
• InfinityLab bio-inert flow paths: PEEK-lined columns, titanium/PEEK capillaries, deactivator additives
• InfinityLab Poroshell 120 HILIC, HILIC-Z (zwitterionic), and HILIC-OH5 chemistries

Main Results and Discussion

HILIC exhibits reversed elution order versus RPLC, releasing analytes from least to most polar. Method development highlights:

• Mobile phases: 5–30 mM volatile buffer in water (pH 3–11) paired with acetonitrile (70–95%) for hydration and retention control
• Column equilibration: ramp aqueous content to accelerate stabilization (e.g., 20–50% water for 25–75 column volumes)
• Sample solvent: maximize acetonitrile proportion, limit injection volume (<2 µL) to avoid peak distortion
• Metal-sensitive assays: apply phosphoric acid passivation, deactivator additive, and PEEK-lined HILIC-Z to minimize metal interactions and improve MS sensitivity

Applications demonstrated include separation of isobaric amino acids (leucine/isoleucine), water-soluble vitamins, organic acids, sugars, and opioid metabolites, often with improved LC–MS sensitivity compared to RPLC.

Benefits and Practical Applications

• Retains cationic, anionic, and neutral polar compounds
• Fully MS compatible for trace-level detection in positive/negative modes
• Simple adoption on existing LC platforms by column swap
• Complementary selectivity to reversed-phase for challenging pharmaceutical, metabolomic, environmental, and food analyses

Future Trends and Potential Applications

Anticipated developments include integration of HILIC in high-throughput metabolomics workflows, further enhancement of bio-inert stationary phases, and automated sample preparation to streamline polar analyte quantitation. New buffer systems and column chemistries will continue to improve selectivity and robustness for next-generation analytical challenges.

Conclusion

HILIC chromatography extends the analytical toolkit by reliably retaining polar analytes and offering seamless MS compatibility. Optimized mobile phase composition, sample solvent management, and column care protocols ensure reproducible performance. Utilizing bio-inert hardware and deactivator additives further broadens HILIC’s utility for metal-sensitive and low-level compound analyses.

Reference

No specific literature references were provided in the source text.